The invention relates to the use of molecular markers in an automated microscope utilizing computer image analysis for biological specimens.
A molecular marker is a chemical that binds with high specificity to a predetermined chemical target inside or on the surface of a cell or to particular molecules suspended in a biological fluid specimen. In common use are molecular markers for proteins, in which case a marker acts as an antibody to a target antigen, and for nucleic acids, in which case a marker is a strand of DNA or RNA with a base sequence complementary to that of the target. Attachment of the antibody to the antigen is called xe2x80x9cbindingxe2x80x9d. Attachment of the nucleic acid probe to its complement is called xe2x80x9chybridizationxe2x80x9d. Automated detection and localization of these attachments and, in some cases, quantification of the amount of target chemical attaching itself to targets, can improve the performance of automated microscopes used to screen specimens for indications of disease. These same methods can also be useful in research for medicine and biology, discovery and testing of pharmaceuticals and in toxicological testing of chemical agents.
The invention applies to molecular markers that utilize light absorbing dyes, also known as chromogens, as opposed to dyes that fluoresce in the presence of excitation or that radiate photons as a product of radioactive decay or chemical reaction. The general approach to staining a specimen with such a marker comprises the steps of concentration, capture, fixation, incubation, and development. Concentration and capture take many forms, but result in the attachment of the cells or molecular components of interest to some surface suitable for examination by the microscope. Fixation lyses the cells in the specimen and preserves them from decay. Incubation exposes the cells or biological fluid to the antibody or nucleic acid molecules, which have been attached to substrate molecules that accomplish the staining. Antigen-antibody binding or nucleic acid hybridization occur during incubation. During development, the substrate undergoes a reaction typically catalyzed by an enzyme to produce an insoluble, light-absorbing precipitate. A further step in the development process includes washing away the unbound marker, leaving precipitate only in those areas where binding or hybridization have occurred.
Chromogens are selected for their light absorption characteristics. Absorption of a particular wavelength produces color at a complementary wavelength. Multiple chromogens can be used on a specimen. Typically, a set of chromogens is used to stain cellular structures for morphological analysis and a single chromogen that yields a distinctive color is used as a molecular marker.
When such biochemical markers are used in conventional microscopy, a trained specialist views the image from the microscope and searches for structures of the color produced by the chromogen of interest. A judgement is then made of the clinical significance of the appearance, if any, of the marker. This judgement may be based on the identification of cell type and degree of morphological abnormality of the cells stained by the marker as well as the concentration of the marker stain.
Therefore, it is one motivation of the invention to provide an automated screening system that uses this information to automatically analyze a biological specimen. It is a further object of this invention to separate molecular marker information from morphological features.
A method for biological specimen image analysis on a plurality of images collected at multiple wavelengths comprising the steps of: applying a chromogen separator to the plurality of images to generate a set of new images; segmenting the each image of the set of new images to provide a plurality of segmented outputs; performing feature calculation on each of the plurality of segmented outputs wherein data from each of the segmented outputs is used on each feature calculation to provide a plurality of feature calculation results; performing object classification on each of the feature calculation results wherein data from each of the feature calculation results is used on each object classification to provide a plurality of object classification results; integrating the object classification results to provide a field of view score integrated output; and generating a slide score from the field of view score integrated output.
Other objects, features and advantages of the present invention will become apparent to those skilled in the art through the description of the preferred embodiment, claims and drawings herein wherein like numerals refer to like elements.